Improvement of Aspergillus oryzae NRRL 3484 by mutagenesis and optimization of culture conditions in solid-state fermentation for the hyper-production of extracellular cellulase

Spore suspensions of Aspergillus oryzae NRRL 3484 were subjected to mutagenesis using ultraviolet-irradiation followed by chemical treatments to improve the biosynthesis of cellulase. Ten mutant strains namely UEAC7, UEAR5, UNAC4, UNAC16, UNAR19, UNBC7, UNBR3, UNBR10, UNBR23 and UNBR25 were selected and their extracellular cellulase activities were assayed. Mutant UNAC4 gave the highest cellulase production [2,455 ± 28 U/g-dry substrate (ds) for filter paper-ase (FP-ase)] in a yield 4-fold exceeding that of the wild type strain (578 ± 5.0 U/g-ds for FP-ase). Rice straw (RS) was used as a sole carbon source for the enzyme production at a concentration of 10 % (w/v). Maximum cellulase production was achieved at initial medium pH 5.5, initial moisture content 77 % and an incubation temperature 28 °C on the fifth day of growth. NH₄Cl proved to be the suitable added nitrogen source for maximum enzyme production followed by peptone. These results clearly indicate the cost-effectiveness of solid state fermentation technology in the economic production of extracellular cellulase. The hyper-production of cellulase by mutant strain UNAC4 has potential for industrial processes that convert lignocellulosic material (e.g. RS) into products of commercial value such as glucose and biofuels.     

Evaluation of cultivar susceptibility and storage periods towards aflatoxin B1 contamination on pistachio nuts

Aflatoxins (AFs) are potent sources of health risks to both humans and animals. Among them, AFB1 is the most hazardously toxic and the most frequent in various food commodities, including pistachio nuts. In this survey, the effect of the storage period on AFB1 accumulation on pistachio nuts was investigated. A total of 49 samples collected during the crop year of 2005 from the most cultivated pistachio cultivars in Tunisia were rapidly screened by enzyme-linked immunosorbent assay (ELISA) combined with an immunoaffinity step. The obtained results showed that the contamination of pistachio nuts has occurred clearly after two years of storage for all the tested cultivars except the case of Mateur variety and Thyna ecotypes. In this study, the cultivar Mateur was found to be the most susceptible cultivar to contamination by AFB1. After 4 years of storage, the average contamination levels in nut samples ranged from 2.7 ± 0.3 to 12.7 ± 2.2 μg/kg for AFB1, according to the cultivar. These levels exceeded the maximum permitted limit of 2 μg/kg set by the European Commission in nuts.     

Exogenous salicylate application affects the lead and copper accumulation characteristics of Lemna gibba L

Previous studies have shown that salicylates can change the ion permeability of root cells. Therefore the possible effects of exogenous salicylate application on lead (Pb) and copper (Cu) accumulation and its protective role against DNA damage due to metal exposure in Lemna gibba were studied. L. gibba was exposed to 5, 10, and 25 microM Pb and Cu for six days in the presence and absence of sodium salicylate (SA) (0.1, 0.5, and 1 mM). At all concentrations tested, SA application decreased Pb accumulation. On the other hand, application of 0.5 mM SA increased Cu accumulation. SA did not reduce DNA damage resulting from Pb and Cu toxicity. In summary, SA may be useful for reducing Pb accumulation, and application of SA at 0.5 mM may be useful for the phytoextraction of Cu.     

Methotrexate binding causes structural and functional changes in lung cystatin

Regulation of cysteine proteinases and their inhibitors is of utmost importance in diseases like lung cancer, chronic inflammatory conditions such as asthma, emphysema, and idiopathic pulmonary fibrosis. Protease-antiprotease imbalance accelerates disease progression. In the present study, the effect of antineoplastic and antirheumatic drug methotrexate (MTX) on lung cystatin (a cysteine protease inhibitor) was studied to explore drug induced changes in functional and structural integrity of the protein. The basic binding interaction was studied by UV-absorption, FT-IR and fluorescence spectroscopy. The quenching of protein fluorescence confirmed the binding of MTX with goat lung cystatin (GLC-I). Stern-Volmer analysis of MTX-GLC-I system at different temperatures indicates the presence of static component in the quenching mechanism. The thermodynamic parameters ΔH? and ΔS? were -3.8 kJ/mol and 94.97 J?mol?1?K?1, respectively, indicating that both hydrogen bonds and hydrophobic interactions played a major role in the binding of MTX to GLC-I. Methotrexate (7 μM) caused complete inactivation of lung cystatin after 6 hours. The results of FT-IR spectroscopy reflect perturbation of the goat lung cystatin on interaction with MTX. Methotrexate induced loss of function change in the inhibitor could provide a rationale for the off target tissue injury caused by the drug and for the design of agents against such an injury.     

Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial beta-amylases

Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.     

Superantigen presentation by airway smooth muscle to CD4+ T lymphocytes elicits reciprocal proasthmatic changes in airway function

Microbial products serving as superantigens (SAgs) have been implicated in triggering various T cell-mediated chronic inflammatory disorders, including severe asthma. Given earlier evidence demonstrating that airway smooth muscle (ASM) cells express MHC class II molecules, we investigated whether ASM can present SAg to resting CD4(+) T cells, and further examined whether this action reciprocally elicits proasthmatic changes in ASM responsiveness. Coincubation of CD4(+) T cells with human ASM cells pulsed with the SAg, staphylococcal enterotoxin A (SEA), elicited adherence and clustering of class II and CD3 molecules at the ASM/T cell interface, indicative of immunological synapse formation, in association with T cell activation. This ASM/T cell interaction evoked up-regulated mRNA expression and pronounced release of the Th2-type cytokine, IL-13, into the coculture medium, which was MHC class II dependent. Moreover, when administering the conditioned medium from the SEA-stimulated ASM/T cell cocultures to isolated naive rabbit ASM tissues, the latter exhibited proasthmatic-like changes in their constrictor and relaxation responsiveness that were prevented by pretreating the tissues with an anti-IL-13 neutralizing Ab. Collectively, these observations are the first to demonstrate that ASM can present SAg to CD4(+) T cells, and that this MHC class II-mediated cooperative ASM/T cell interaction elicits release of IL-13 that, in turn, evokes proasthmatic changes in ASM constrictor and relaxant responsiveness. Thus, a new immuno-regulatory role for ASM is identified that potentially contributes to the pathogenesis of nonallergic (intrinsic) asthma and, accordingly, may underlie the reported association between microbial SAg exposure, T cell activation, and severe asthma.     

Vinpocetine ameliorates acute hepatic damage caused by administration of carbon tetrachloride in rats

Vinpocetine is a widely used drug for the treatment of cerebrovascular and memory disorders. This study aimed to investigate the effect of vinpocetine on the acute hepatic injury caused in the rat by the administration of CCl4 in vivo. Vinpocetine (2.1, 4.2, 8.4 mg/kg) or silymarin (30 mg/kg) was given once daily orally simultaneously with CCl4 and for 15 days thereafter. Liver damage was assessed by determining serum enzyme activities and hepatic histopathology. Stained sections were subjected to morphometric evaluation using computerized image analyzer. The results showed that vinpocetine administered to CCl4-treated rats decreased the elevated alanine aminotransferase (ALT) by 49.3, 58.1 and 63.6%, aspartate aminotransferase (AST) by 10.5, 22.6 and 27.2% and alkaline phosphatase (ALP) by 52.5, 59.6 and 64.9%, respectively, and in a dose-dependent manner. Meanwhile, silymarin reduced elevated ALT, AST and ALP levels by 53.1, 26.9 and 66%, respectively. Histological examination of liver specimens revealed a marked reduction in liver cell necrosis in vinpocetine and silymarin-treated rats compared with vehicle-treated CCl4-treated rats. Quantitative analysis of the area of damage showed 85.3% reduction in the area of damage after silymarin and 72.2, 78.9 and 82.6% reduction after vinpocetine treatment at 2.1, 4.2, 8.4 mg/kg, respectively. It is concluded that administration of vinpocetine in a model of CCl4-induced liver injury in rats reduced liver damage. The reduction obtained by 4.2 mg/kg of vinpocetine was similar to that obtained by 30 mg/kg silymarin. Therefore, it is suggested that vinpocetine might be a good pharmacological agent in the treatment of liver disease besides its neuroprotective effects.